It is increasingly recognized that the mammalian DNA not only acts as static storage of genetic information, but also is subject to dynamic and reversible epigenetic modifications at cytosine bases by a highly orchestrated cascade of enzymatic activities. Beyond four bases (A, T, C and G) of genetic code, there are four modified cytosine bases (5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5- carboxylcytosine (5caC)) present in the mammalian genome. 5mC pattern is first established by DNA methyltransferases (DNMTs) and TET family of DNA dioxygenases can then convert 5mC to 5hmC, 5fC and 5caC via successive oxidation reactions. 5fC and 5caC can be further excised and repaired to regenerate cytosine by Thymine DNA glycosylase (TDG) and base excision repair (BER) pathway. Mutations in many enzymes involved in catalyzing specific cytosine modification are implicated in a variety of human diseases including cancers and neurological disorders. Despite their critical roles, efforts to decipher the mechanism and function of dynamic changes in cytosine modification states are largely impeded by the lack of technologies to accurately determine the location of all 5mC oxidation derivatives (5hmC/5fC/5caC) and methods to functionally interfere this dynamic process at specific loci. This application describes a combination of epigenomics, bioinformatics and synthetic biology approaches to advance the understanding of in vivo function of TET/TDG-mediated active DNA demethylation in mammalian cells. First, to overcome the limitation in resolution associated with antibody-based affinity enrichment method, I will explore the biochemical expertise in the Zhang lab and develop new genome-wide technologies capable of mapping of 5fC/5caC at a single-nucleotide resolution. In addition, I will learn to construct effective computational pipeline to analyze these base-resolution mapping datasets. Second, site-specific transcription activator-like effector (epiTALEs) fused with catalytic domains of epigenetic enzymes will be constructed to investigate causal effect of TET/TDG activity on regulatory functions of gene regulatory elements. Lastly, I will take advantage of the new technologies and computational pipelines developed in Drs. Yi Zhang (Boston Children's Hospital/Harvard Medical School) and Shirley Liu laboratories (Harvard School of Public Health/Dana Farber Cancer Institute) as well as stem cell/cardiovascular biology expertise learned in Dr. Chien laboratory (Harvard University) during my mentored phase to study the role of the cytosine modifying epigenetic pathway during lineage differentiation (neural and cardiovascular) and in disease models. This career development award will allow me to develop new technologies and further strengthen my computational skills. Combining these new skills with my previous training in epigenetics and genomics will better prepare me as an independent investigator. The excellent research environment in Boston Children's Hospital and Harvard Medical School will greatly facilitate my research and career development during both mentored phase and my transition to an independent academic position. Collectively, the proposed study will pave the road to launch my future research that aim at elucidating the rules governing dynamic regulation of DNA methylation in normal physiological processes and human diseases.